Inhibition of Urease from Seeds of Water-melon (Citrullus vulgaris) by Heavy Metal Ions

Urease from the seeds of water melon was found to be inhibited by heavy metal ions like copper, lead, nickel and cobalt. The order of effectiveness of these metals as inhibitors was Cu²⁺ > Pb²⁺ > Ni²⁺ > Co²⁺. The inhibition by these ions was noncompetitive. Time — dependent interaction of urease with nickel and cobalt exhibited a biphasic inhibition behaviour in which approximately half of the initial activity was lost rapidly (within 2 min) and remainder in a slow phase. The inhibition was largely irreversible, hence could not be reversed by dialysis. These observations are suggestive of half-and-half distribution of — SH groups on the native enzyme resulting urease into asymmetric oligomeric molecule.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Molecular Asymmetry in Urease of Water Melon (Citrullus vulgaris)

Kinetics of urease-catalysed urea hydrolysis follows Arrhenius equation in the temperature range 10-50°C and shows an energy of activation equal to 7.14 kcal/mol. The kinetics of thermal inactivation of the enzyme is biphasic, In that half of the initial activity is destroyed more rapidly than the remaining half. The data are consistent with the rate equation: A_t = A_fast·e^-k _fast ^-t + A_slow ·e^-K _slow ^-t where A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. A similar activity decay (namely blphaslc) is also observed on storing the enzyme at +4 and ?4OC. The data suggest the existence of half-and-half distribution of sites which is a manifestation of a pre-exlstent site heterogeneity in the oligomeric protein molecule.     

The Interrelationship of Canavanine and Urease in Seeds of the Lotoideae

Seeds of 29 species of canavanine-synthesizing legumes wereassayed for their urease and canavanine production. All of theexamined species possess detectable urease activity. In general,the leguininous seeds richest in urease also had the most canavanine. The urease content of the jack bean seed, Canavalia ensiformis(L.) DC., is formidable and disproportionally greater than thequantity of stored canavanine. The massive urease content ofthe seed cannot be rationalized by the magnitude of the canavaninepool. Analysis of eight species of Mucuna demonstrated that canavanineis not stored in the seeds of these plants. Mucuna species donot appear to be unique in having seeds that do not concurrentlyproduce urease and canavanine.     

Inhibition of Water Melon (Citrullus vulgaris) Urease by Fluoride

Effect of fluoride on the activity of purified urease from seeds of watermelon (Citrullus vulgaris) was studied. Fluoride exhibited a concentration dependent inhibition both in presenceand absence ofthe substrate. The inhibition was non-competitive. Addition of 8mM β-mercaptoethanol gradually abolished the fluoride inhibition. β-mnercaptoethanol, in presence of fluoride, also exhibited a concentration dependent suppression of inhibition caused by fluoride. The significance of these observations is discussed.     

Effect of Water Stress on the Carbohydrate Metabolism of Citrullus lanatus Seeds during Germination

Gluconeogenesis in Citrullus lanatus seeds is a post germinative event. Increases in isocitrate lyase activity and incorporation of radioactivity from [2-¹⁴C]acetate into sugars occur only after radicle emergence. During germination, the seeds appear to rely on carbohydrate as the respiratory substrate. At this time, glycolysis, the pentose phosphate pathway, and the tricarbocyclic acid cycle seem to be functional. Utilization of raffinose during germination appears to be important.

Water stress, which completely inhibits germination, has a marked effect on carbohydrate metabolism. The rate of ¹⁴CO₂ release from [2-¹⁴C]acetate, [1-¹⁴C]glucose, and [6-¹⁴C]glucose is lower in the stressed seeds than the control seeds during the respiratory lag phase. However, in the stressed seeds neither glycolysis, the pentose phosphate pathway, nor the tricarboxylic acid cycle is completely inhibited. In contrast to the control seeds in which raffinose content sharply declines after 12 h of incubation, raffinose content in the stressed seeds remains fairly constant.

The respiratory lag phase of the control seeds coincides with a lower reducing substance content, glucose content, and fructose content than in the stressed seeds during the corresponding incubation period.

     

幽门螺杆菌尿素酶抗原的分离及纯化

本实验采用新型离了交换剂Ｓｏｕｒｅｃｅ Ｑ１５为分离介质,用氯化钠盐浓度梯度洗脱法,从幽门螺杆菌超声处理上清液中纯化了幽累相菌尿毒酶抗原,所得纯化物经ＳＤＳ－ＰＡＧＥ电泳分析证明具有良好的纯度,只含有分子量为６６０００及２９５００两种蛋白成分,与幽门螺杆菌两种亚基的大小安全相同。以此纯化尿素酶为抗原用于ＥＬＩＳＡ检测临床确诊的幽门螺杆菌感染者血清１２全水感染幽门螺杆者血清１８例,其ＥＬＩＳＡ阳性及     

Genome Sequencing of up to 6,000-Year-Old Citrullus Seeds Reveals Use of a Bitter-Fleshed Species Prior to Watermelon Domestication

Iconographic evidence from Egypt suggests that watermelon pulp was consumed there as a dessert by 4,360 BP. Earlier archaeobotanical evidence comes from seeds from Neolithic settlements in Libya, but whether these were watermelons with sweet pulp or other forms is unknown. We generated genome sequences from 6,000- and 3,300-year-old seeds from Libya and Sudan, and from worldwide herbarium collections made between 1824 and 2019, and analyzed these data together with resequenced genomes from important germplasm collections for a total of 131 accessions. Phylogenomic and population-genomic analyses reveal that (1) much of the nuclear genome of both ancient seeds is traceable to West African seed-use “egusi-type” watermelon (Citrullus mucosospermus) rather than domesticated pulp-use watermelon (Citrullus lanatus ssp. vulgaris); (2) the 6,000-year-old watermelon likely had bitter pulp and greenish-white flesh as today found in C. mucosospermus, given alleles in the bitterness regulators ClBT and in the red color marker LYCB; and (3) both ancient genomes showed admixture from C. mucosospermus, C. lanatus ssp. cordophanus, C. lanatus ssp. vulgaris, and even South African Citrullus amarus, and evident introgression between the Libyan seed (UMB-6) and populations of C. lanatus. An unexpected new insight is that Citrullus appears to have initially been collected or cultivated for its seeds, not its flesh, consistent with seed damage patterns induced by human teeth in the oldest Libyan material.     

Agrobacterium-mediated transformation in Citrullus lanatus

Agrobacterium tumefaciens-mediated transformation was used to produce transgenic watermelon. Cotyledonary explants of Citrullus lanatus Thumb (cv. Daesan) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing pPTN289 carrying with bar gene and pPTN290 carrying with nptII gene, respectively. There was a significant difference in the transformation frequency between bacteria strains and selective markers. The EHA101/pPTN289 showed higher transformation frequency (1.16 %) than GV3101/pPTN289 (0.33 %) and LBA4404/pPTN289 or /pPTN290 (0 %). The shoots obtained (633 and 57 lines) showed some resistance to glufosinate and paromomycin, respectively. Of them, the β-glucuronidase positive response and PCR products amplified by bar and nptII specific primers showed at least 21 plants resistant to glufosinate and at least 6 plants to paromomycin. Southern blot analysis revealed that the bar gene integrated into genome of transgenic watermelon. Acclimated transgenic watermelons were successfully transplanted in the greenhouse and showed no phenotypic variation.     

脲酶抑制剂氢醌对土壤脲酶活性,氨挥发和硝化的抑制动态

1.氢醌对土壤脲酶活性的抑制率及其持续的时间同氢醌浓度成正相关,与土壤脲酶活性成负相关。2.氢醌能有效地抑制施入土壤中尿素氨的挥发,而对铵盐和尿素的硝化强度产生强烈抑制。3.在麦秸还田土壤中,由于脲酶活性增高而提高了施入尿素的水解速度,故需提高氢醌用量;但由于麦秸的“氮因子效应”又固定了尿素分解产物及其氧化产物,从而弥补了氢醌失效后可能造成氮素的继续损失。     

Kinetics of Thermal Inactivation and Molecular Asymmetry of Urease from Dehusked Pigeongea (Cajanus cajan L.) Seeds

The purified urease from pigeonpea was moderately stable at ?10°C. The shelf-life of the enzyme on storage in 0.1 M Tris-acetate buffer, pH 6.5, at ?10°C showed a single exponential decay with a t_1/2 of approx. 30 days. In the presence of additives like 5mM dithiothreitol the t_1/2 increased to 223 days at the same temperature, in a single exponential decay. The Arrhenius plot of the kinetics of the pigeonpea urease catalysed urea hydrolysis over the temperature range 27 to 77°C, was linear. The Q₁₀ value was found to be 1.46. The energy of activation calculated from the Arrhenius equation was found to be 5.1 kcal/mole active site. The thermal denaturation of pigeopea urease at 65 and 70°C was found to obey biphasic kinetics in which half of the activity is destroyed faster than the remaining half. The time course of thermal inactivation can be described by the following equation, consisting of two first order terms: A_t = A_fast.e^-k _fast + A_slow.e ^-kslow^.t where, A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. The data suggests the existence of site-site heterogeneity in oligomeric urease molecule from pigeonpea.     

Urease activity in soils

Summary The availability of Ca from different levels of gypsum and calcium carbonate in a non-saline sodic soil has been investigated. Different levels of tagged gypsum (Ca⁴⁵SO₄.2H₂O) and calcium carbonate (Ca⁴⁵CO₃) (i.e. 0, 25, 50, 75, and 100 per cent of gypsum requirement) were mixed thoroughly in 3.5 Kg of a non-saline alkali soil (ESP, 48.4; ECe, 2.68 millimhos/cm). Dhaincha (Sesbania aculeata) — a legume and barley (Hordeum vulgare L.) — a cereal were taken as test crops. Increasing levels of gypsum caused a gradual increase in the yield of dry matter, content of Ca and K in the plant tops and Ca:Na and (Ca+Mg):(Na+K) ratios in both the crops. Application of calcium carbonate caused a slight increase in the dry matter yield, content of Ca and Mg and Ca:Na and (Ca+Mg):(Na+K) ratios in barley, however, in case of dhaincha there was no such effect. Gypsum application caused a gradual decrease in the content of Na and P in both the crops. Total uptake of Ca, Mg, K, N and P per pot increased in response to gypsum application. The effect of calcium carbonate application on the total uptake of these elements was much smaller on dhaincha, but in barley there was some increasing trend.Increasing application of tagged gypsum and calcium carbonate caused a gradual increase in the concentration and per cent contribution of source Ca in both the crops, although, the rate of increase was considerably more in dhaincha. The availability of Ca from applied gypsum was considerably more than that from applied calcium carbonate. Efficiency of dhaincha to utilize Ca from applied sources was considerably more (i.e. about five times) than that of barley     

Fat Metabolism in Germinating Citrullus vulgaris

A column chromatographic technique, enabling identificationand quantitative estimation of fatty acids, has been employedto study fat metabolism in Citrullus during germination in thelight. This plant is characterized by an unusually rapid disappearanceof storage fat as the cotyledons expand and turn green. In spiteof the high catabolic activity there is no evidence for accumulationof free fatty acids or short-chain fatty acids at this stage.Information on this point derived from acid value or saponificationvalue of the oil is shown to be untrustworthy. Citrullus seed fat contains the following percentages of acids:linoleic 70·6, oleic 7·2, palmitic 10·1,stearic 11·2, and arachidic 0·6, and careful analysishas also revealed small amounts of octadecatrienoic acids, bothconjugated and non-conjugated. All the major acids are brokendown at rates proportionate to the quantities originally present,with the exception of oleic acid which is metabolized somewhatmore rapidly. ‘Linolenic’ acid is synthesized in the expandinggreen cotyledons and the fatty acid composition of the latter,in the late germination stages, resembles that of a green leafand is very different from that of the seed. The results suggest a rapid removal of storage fat from thecotyledons and concomitant formation in small quantity of atypial leaf fat as the new photo-synthetic function develops.     

脲酶在生物工程中的应用

脲酶是一种高效的尿素分解催化剂,化学反应速度是常规化学催化的1014倍,广泛应用于工业、农业和医药行业.论述脲酶在生物工程中的应用,内容包括检测血、尿、酒饮料、天然水和环境污水中尿素;检测肌酐和精氨酸;消除肾衰竭病人体内多余尿素,消除酒饮料和废水中尿素;回收太空飞船上的废水和控制多级酶促反应中的pH.     

交联脲酶聚集体的制备和初步应用

为提高游离脲酶的稳定性,将游离脲酶用硫酸铵沉淀下来后,以戊二醛作为交联剂对其进行化学交联,制备新型的固定化脲酶交联脲酶聚集体,并对其酶学性质进行研究。交联脲酶聚集体的最适pH、最适温度和K_m值分别为：pH 8.0、70℃和0.021 mol/L。在对交联脲酶聚集体的热稳定性,储存稳定性,和对抗蛋白水解酶的能力的研究中,交联脲酶聚集体均显示了比游离脲酶更高的稳定性。为考察其使用效果和稳定性,将其与包醛氧淀粉联合,用于慢性肾衰动物模型的口服治疗。以腺嘌呤灌胃法(每天300mg/kg, 共30d)制备慢性肾衰动物模型,将23只大鼠随机分为模型对照组(每天以10mL/kg蒸馏水灌胃)、单纯包醛氧淀粉组(给予含包醛氧淀粉饲料,10mL/kg蒸馏水灌胃)和包醛氧淀粉+交联脲酶聚集体组(给予含包醛氧淀粉饲料,交联脲酶聚集体悬浮液10mL/kg灌胃),经2周治疗后, 模型对照组、治疗对照组和治疗组实验前后的肌酐含量均有小幅下降,但差异不显著(P值分别为0.922、0.972和0.225＞0.05)。模型对照组的尿素氮含量变化不明显(P=0.211＞0.05)。治疗对照组和治疗组实验前后的尿素氮含量均明显下降(P值分别为0.004和小于0.001,均小于0.01)。治疗对照组和治疗组实验前后的尿素氮含量下降量比较,有显著性差异(P=0.016＜0.05)。治疗组尿素氮含量下降更明显。说明交联脲酶聚集体和包醛氧淀粉联合使用时,对尿素的清除效率比单纯使用包醛氧淀粉更高。     

Purification and Properties of Urease Derived from Hydrated Seeds of Jack Bean, Canavalia ensiformis (L) DC

Urease from jack bean meal and hydrated seeds has been obtained in 25 to 33% yield with specific activity in the range of 1000 to 1070 units/mg protein. A purification of 100 to 130-fold was achieved from meal and fully soaked seeds. Use of β-mercaptoethanol and EDTA was found essential to obtain this high yield and purity. Amino acid analysis showed all 18 amino acids commonly found in proteins. Electrophoresis of urease from soaked seeds (specific activity: 1025 units/mg protein) on a starch-gel block showed 2 peaks. Upon ultracentrifugation of urease samples having a low specific activity (less than 25% pure), the major portion of the urease was probably present in a peak having a sedimentation value of 11 to 12. With relatively pure samples (55-100% pure). S values in the range of 18 to 20 and 24 to 26 were obtained. Usually the purest samples of urease tested without any prior storage lacked the 24 to 26 S peak or the higher polymeric forms. The percentage areas under none of the ultracentrifuge peaks corresponded to the percentage purity of the sample analyzed. It is argued that the physical state of urease in the cell when associated with other seed proteins is as yet uncertain. In crude extracts, a portion of urease exists in a 12 S form but so far data on its origin and specific activity in relation to other species of urease are not available.